mmg_233_2013_genetics_genomicswikiaorg-20200214-history
TfR1/TfR2
]Transferrin (Tf) is an iron binding protein that helps regulate free iron levels in the body. Each Tf molecule can bind to two ferric iron ions. Once bound to iron, Tf is recognized by it's receptor TfR, which is able to bind two Tf molecules. After Tf binds to TfR, it is internalized in the cell through receptor mediated endocytosis. Following endocytosis the pH in the endocytosed vacuole drops, and causes iron to be released. The two Tf molecules, still bound to the TfR, are the recycled to the cell surface where they are then released by TrR to undergo another cycle or iron binding. Defects in iron binding and transport can lead to the development of hemochromatosis, which is characterized by a decrease in cellular uptake of iron leading to deposits of iron in the body. The protein HFE binds to the TfR in competition with Tf and reduces the amount of cellular iron uptake.2 In the study discussed below, researchers examined how HFE binds to the TfR using chimeric TfR constructs in an effort to better understand the mechanisms of iron storage in the body. Recombinant protein In this study chimeric constructs of TfR1 and TfR2 were created to help determine what domains were involved in HFE binding. (See figure) The cytoplamsic domains (cd) transmembrane domains ™ and ecto-domain (ecto) were interchanged. Additionally they created chimeric HFE constructs to assess what domains were involved in protein binding interactions. The TfR and HFE constructs were created using several different plasmids containing differnet pcDNA3.1/TfR sequences. Once the plasmids were created they were stably transfected into HeLa cells for use in experiments.3 Using these chimeric domains is useful in the study of protein binding because it allows you to determine more accurately what regions of the proteins are interacting, and can give more detailed findings as to what components are important in iron uptake. Use in this study Proteins were purified using an immunoprecipitation or affinity purification. Some of the constructs contained HA tags or FLAG tags, which were used to help purify proteins. This was then helpful in determining protein domain interactions though identification of proteins that would coIP. It has previously been shown that the helical domain of TfR1 binds to the a1 and a2 helices of HFE, however it was unknown what binding sites were used by TfR2. This study used the chimeric TfR construdts to see if TfR2 binds in a similar manner to TfR1. It was found that TfR2 binds to HFE different from TfR1, by interacting with the HFE a3 domain and functions independently from TfR1. References 1 Figure 1. Yifan Cheng, Olga Zak, Philip Aisen, Stephen C. Harrison, Thomas Walz. Single particle reconstruction of the human apo-transferrin–transferrin receptor complex. Journal of Structural Biology, Volume 152, Issue 3, December 2005, Pages 204–210 http://dx.doi.org/10.1016/j.jsb.2005.10.006 2 The Molecular Pathogenesis of Hereditary Hemochromatosis. Jodie L. Babitt, M.D.,1 and Herbert Y. Lin, M.D., Ph.D. Semin Liver Dis. 2011 Aug;31(3):280-92 3 Chen J, Chloupková M, Gao J, Chapman-Arvedson TL, Enns CA.HFE Modulates Transferrin Receptor 2 Levels in Hepatoma Cells via Interactions That Differ from Transferrin Receptor 1-HFE Interactions J Biol Chem. 2007 Dec 21;282(51):36862-70